L-carnitine (3-hydroxy-4-trimethylamino-butyrate), which is also known as vitamin BT, is a natural vitamin analog that is very important in human metabolism. L-carnitine was originally isolated from bovine muscle tissue in 1905 by two Russian scientists, Gulewitsch and Krimberg, and its chemical structure was identified in 1932. L-carnitine is found in nearly all cells of the body and transports activated free long-chain fatty acids across the inner membrane of the mitochondria. Since the inner mitochondrial membrane is an impenetrable barrier to acyl-CoA esters, free long-chain fatty acids, activated to acyl-CoA esters in the cytoplasm, pass across the membrane when esterified to L-carnitine. When L-carnitine is present in low levels in the skeletal muscles, liver, heart and kidneys, free long-chain fatty acids are difficult to utilize as an energy source. This abnormal carnitine metabolism causes various diseases, including growth retardation, cardiomyopathy and muscle weakness. When L-carnitine is not synthesized in suitable amounts in the body, carnitine should be absorbed from foods to avoid carnitine deficiency symptoms. Especially in infants who are not able to biosynthesize L-carnitine, L-carnitine is an essential nutrient.
L-carnitine is used as an active component in pharmaceutical preparations. Exogenous supplementation of L-carnitine is required to treat carnitine deficiency and other diseases, especially cardiac diseases. Recently, this therapeutic use of L-carnitine has become increasingly important (R. A. Frenkel and J. D. Mc Garry; “Carnitine biosyntheis, metabolism and functions”, Academic Press, 1980).
L-carnitine has been identified as playing many important roles in the body. However, conventional methods including biological extraction are not suitable for mass production of L-carnitine. One method capable of easily obtaining L-carnitine is to utilize DL-carnitine including optical isomers. This method causes side effects in the body because it contains D-carnitine (Curr. Ther. Res. 28, 195-198, 1980). In many cases, D-carnitine competes with L-carnitine in the body and interrupts the mitochondrial beta-oxidation of free long-chain fatty acids. In patients having remarkably reduced renal function, this impaired metabolism of long-chain fatty acids leads to more serious inhibition.
Many efforts have been made to obtain optically pure L-carnitine, which include a chemical optical resolution method (U.S. Pat. No. 5,166,426), a biological method using microorganisms or enzymes (U.S. Pat. No. 5,187,093), and a method of producing L-carnitine using a chiral compound as a starting compound (U.S. Pat. No. 6,420,599 B2).
Among various methods for obtaining L-carnitine, a biological method using microorganisms or enzymes employs a biological enzyme, gamma-butyrobetaine hydroxylase, to produce optically active L-carnitine. This enzyme was isolated in mice and humans (Rebouche and Engel, J Biol Chem 255:8700-8705, 1980), and its nucleotide sequence was identified. Higher organisms including mammals utilize an amino acid residue of proteins, lysine, as a precursor for L-carnitine biosynthesis, whereas Neurospora crassa produces optically pure L-carnitine from free lysine (Fraenkel, Biol Bull, 104:359-371, 1953). The mechanism of L-carnitine biosynthesis is briefly as follows. Carnitine synthesis begins with methylation of lysine by S-adenosylmethionine acting as a methyl donor, resulting in the formation of ε-N-trimethyllysine. Trimethyllysine is enzymatically transformed into β-hydroxy-trimethyllysine. From the synthesized β-hydroxy-trimethyllysine, trimethylaminobutyl aldehyde is formed, and is then converted to γ-butyrobetaine.
A nucleotide sequence encoding γ-butyrobetaine hydroxylase, which is derived from Neurospora crassa and produces L-carnitine using γ-butyrobetaine, produced through the aforementioned mechanism, as a precursor, has not been identified prior to the present invention.